Corneal imaging is useful in assessing corneal health, monitoring the progression of corneal disease, and evaluating the efficacy of corneal treatments. A corneal confocal microscope is an example of an imaging device that measures characteristics of the cornea. In vivo confocal microscopy allows for high resolution, reliable, real-time imaging of the living corneal microstructure to evaluate, for example, normal corneal morphology, pathogen invasion, dystrophies and degenerations, post surgical management, dry eyes, drug toxicities, endothelial monitoring, and contact lens related changes.
A normal, healthy cornea is a transparent, avascular connective tissue made up of five layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. The corneal epithelium, the outermost layer of the cornea, is a tissue having a thickness of about 50 μm composed of 5 to 6 layers of cells. The corneal epithelium represents about one-tenth of the thickness of the cornea. The corneal epithelium can be divided into three anatomical groups: superficial cells, wing cells, and basal cells.
Superficial epithelial cells are flat polygonal cells that are stacked two to three cell layers deep on the outermost surface of the cornea. When imaged, these cells are characterized by a polygonal pattern, bright illuminated cytoplasm, a reflective nucleus and a perinuclear dark halo. As cells die, the entire cytoplasm becomes hyper-reflective. These superficial cells are up to 50 μm in diameter and about 5 μm thick. They are typically least dense in the corneal center, at around 624 cells/mm2, and typically most dense in the periphery, at around 1213 cells/mm2.
Immediately under (i.e., posterior to) the superficial cells are the wing cells. Wing cells are two to three cells deep. They can be divided into upper (larger) and lower (smaller), but are generally around 20 μm in size and form a regular mosaic pattern. The average density is 5000 cells/mm2 in the central cornea and 5500 cells/mm2 in the periphery.
The inner most layer (i.e., most posterior) of epithelial cells is the basal epithelium. These are the smallest of the epithelial cells, averaging around 8-10 μm. When imaged, they appear as a dense mosaic with highly reflective borders (tight junctions). The average density varies from 6000 to 9000 cells/mm2 in the center and greater than 10,000 cells/mm2 in the periphery.
The sub-basal nerve plexus is immediately adjacent to the basal epithelium. When imaged, the nerve plexus is seen as a relatively cell-free layer with parallel linear hyper-reflective fibers. The nerves are characterized by local axon enlargements which are accumulations of mitochondia and glycogen particles. The fibers are organized into a vortex pattern and therefore will run in different directions depending on the scan location.
Bowman's layer is 8-10 μm thick and consists of randomly arranged collagen fibrils located between the basal cell layer and the stroma. This layer often appears hazy and dysmorphic.
The stroma takes up 80-90% of the whole corneal volume. It consists of cellular, acellular and neurosensory structures. The cellular component (keratocytes) has reflective nuclei, whereas the acellular component (collagen lamellae) appears black or optically transparent. Keratocyte density is highest in the anterior-stroma, declines in the mid-stroma and increases slightly again towards the posterior-stroma. Stromal nerve fibers are thicker than sub-epithelial nerve fibers.
Descemet's membrane may not be visible using confocal microscopy.
The endothelium is a single layer of cells which form a hexagonal mosaic pattern. Healthy endothelium consists of 2500-3000 cells/mm2, however, this decreases with age, disease, and low-oxygen transmissible contact lens wear.
Immune cells, including leukocytes, protect against foreign invaders. The main categories of leukocytes include granular (e.g., neutrophils, basophils and eosinophils), non-granular (e.g., macrophages) and lymphocytes. Granulocytes are typically very small (<10 μm) highly motile and readily invade the cornea during inflammation in response to chemotaxic factors from microbes and injured cells. Macrophages (up to 20 μm) are typically present at the ulcer site and may remain for many months within the tissue. Lymphocytes are found in the palpebral and tarsal conjunctiva. Leukocytes are typically located at the level of the basal or wing cells. Though they are not easily differentiated by confocal microscopy, location, size, and morphology may aid in identification. For example, immune cells may generally migrate along the nerve plexus. They may also, for example, be identified in the basal epithelium and Bowman's layer.
A confocal microscope works by measuring light reflected within a clear or opaque tissue. A corneal confocal microscope illuminates a small region of the cornea with a collimated light source that passes through an aperture and is focused through an objective lens to a tiny volume of space at the focal region of the lens. Reflected light from the focal region is then recollected by the objective lens. The light then passes through a beam splitter and a pinhole before entering a photodetection apparatus. The detector aperture blocks scattered light, resulting in sharper images than those from conventional light microscopy techniques. The photodetection device transforms the light signal into an electrical one, creating a digital histological image.
In vivo confocal microscopy typically has been used clinically to evaluate various corneal pathologies, including infectious keratitis (in particular, Acanthamoeba and fungal keratitis), corneal dystrophies, and other parameters of corneal health and disease. However, in vivo confocal microscopy may yield images containing a massive amount of data that may be difficult to analyze and interpret consistently and quickly. Therefore, most applications of in vivo confocal microscopy have been qualitative or have required time-consuming manual analysis to yield quantitative results. Consequently, there is a need in the art for robust and rapid image processing techniques to objectively evaluate confocal microscopy images to quantify corneal changes.